The present invention concerns a simple and sensitive assay for measurement of adenosine in small samples using definite concentrations of reduced S-adenosylhomocysteine (SAH) hydrolase and radioactive adenosine. The SAH hydrolase-adenosine complex is separated preferably by a filtration equipment and the radioactive adenosine incorporated in this SAH hydrolase-adenosine complex is measured by scintillation counting.
A further objective of the invention is the combination of the analytical substances required for the assay.
Adenosine is an endogenous nucleoside that interacts in various physiological processes which are also regulated by hormones and neurotransmitters. Adenosine actions are mediated by stimulation of specific cell surface receptors which are found to be ubiquitous in mammals.
In the cytosol hydrolysis of AMP leads to adenosine by the activity of endo-5xe2x80x2-nucleotidase. Another source of adenosine generation is the hydrolysis of SAH to adenosine and homocysteine by SAH hydrolase. Adenosine, a product of the energy metabolismn, functions as a mediator in the metabolic control of organ function in heart, kidney and brain. Current research in biochemistry, pharmacology, physiology and clinical chemistry is carried out with the aim to investigate the physiological role and therapeutical potency of adenosine and several adenosine derivates in terms of treating diseases in which adenosine metabolism is deranged and thus organ function impaired. The methods available for measurement of adenosine are:
1. Photometrical Detection
The reduction of adenosine concentration is measured photometrically at 265 nm in the presence of adenosine deaminase. The disavantage of this method is the requirement of sample purification prior measurement to remove purine compounds and other endogenous substances. The detection limit for adenosine is 10xe2x88x927 mol/l.
2. High pressure Liquid Chromatography (HPLC)
The HPLC method combines separation of adenosine and its photometrical detection of adenosine at 254 nm. This method, however, has its limitation with respect to sufficient separation of several interfering nucleotides. In addition, the sensitivity to detect adenosine at 0.3 xcexcmol/l is to low for small samples volume.
3. Radioimmunoassay
This method is based on an antibody method with adenosine 2xe2x80x2, 3xe2x80x2-O-disuccinyl-3-[125I]-Iodtyrosin methyl ester as tracer and with an antibody against disuccinylated-adenosine. For this method a sample purification is not required but adenosine in each sample must be coupled to 2xe2x80x2,3xe2x80x2-O-disuccinyl-3-[125I]-Iodtyrosin. The detection limit is more sensitive (6xc2x7108 mol/l) when compared with the photometric or HPLC methods.
The subject of the invention is correspondingly a sensitive method suitable for detection of adenosine in small sample of 10 xcexcl or biopsies of 2-10 mg tissue without purification of deproteinized samples.
The subject of the invention was resolved by an assay for measurement of adenosine in small samples using reduced SAH hydrolase and radioactive adenosine in definite concentrations. The SAH hydrolase-adenosine complex was separated preferably by a filtration equipment e.g. filtration apparatus for separation of reaction mixtures (DE 197 49 929 A1). The radioactivity incorporated in this complex was measured by scintillation counting.
The further subject of the invention is the largely improved ability of the reduced form of the enzyme SAH hydrolase to bind adenosine with high affinity.
The following table 1 demonstrates that the reduced enzyme is able to bind adenosine with high affinity. The table 1 also discloses that several substances that bind to the active form of the enzyme have a much lower affinity to the reduced form of the enzyme.
The particular advantage of the new invention is that the reduced enzyme retains its ability to bind adenosine with high affinity (KD2xc2x710xe2x88x928 mol/l). Since interferences of endogenous substances with the binding of radioactive adenosine are no longer present, no sample purification is necessary to measure adenosine in deproteinized samples.
The following example is given for the purpose of illustrating the invention:
SAH hydrolase is purified with classical chromatographical methods to homogeneity from organs e.g. from bovine kidney. Fresh bovine kidney (450 g), obtained from the local slaughterhouse were homogenized in two volumes of 50 mM potassium phosphate buffer pH 7.0, 1 mM DTT, 1 mM EDTA and 10 mM PMSF. The homogenate was centrifuged at 20000xc3x97g for 60 min. Ammonium sulfate (164 g/l) was added to the supernatant. After centrifugation at 20000xc3x97g for 30 min the precipitate was disolved in 300-400 ml 20 mM Tris/HCl pH 7.5, 1 mM DTT and 1 mM EDTA and dialyzed against the same buffer.
Chromatography
1. DEAE Sepharose Fast Flow
The dialyzed enzyme solution was applied onto a column (5xc3x9725 cm) of DEAE Sepharose(copyright) Fast Flow equilibrated with the dialysis buffer. The column was washed with 1700 ml of the same buffer and the enzyme was eluted by a 2000 ml linear gradient of 0 to 0.4 M KCl in 20 mM Tris/HCl pH 7.5, 1 mM DTT and 1 mM EDTA. Active fractions were eluted between 250 and 350 mM KCl (flow rate 10 ml/min) pooled and dialyzed against 50 mM potassium phosphate buffer pH 6.8, 1 mM DTT and 1 mM EDTA.
2. Hydroxylapatite
The dialyzed enzyme solution was then applied onto a column of hydroxylapatite (5xc3x9710 cm) equilibrated with 50 mM potassium phosphate pH 6.8, 1 mM DTT, 1 mM EDTA. SAH hydrolase remained in the unadsorbed fractions, which were combined and dialyzed against 10 mM potassium phosphate buffer pH 6.8 (flow rate 1.5 ml/min).
Aminohexyl Sepharose
The dialyzed fractions from the previous step were applied onto a EAH-Sepharose 4B column (2.6xc3x9710 cm) equilibrated with 10 mM potassium phosphate buffer pH 6.8. The column was washed with 200 ml of the same buffer, and the enzyme was eluted by a linear gradient of 0 to 0.4 M KCl in 10 mM potassium phosphate buffer pH 6.8. The SAH hydrolase fractions were eluted between 120 and 200 mM KCl, were pooled and concentrated by an AMICON protein concentrater to a small volume of 3-5 ml.
Superdex(trademark) 200 Gelfiltration
The concentrated SAH-hydrolase solution was chromatographed on a column of Superdex(trademark) 200 (2.6xc3x9760 cm) equilibrated with phosphate buffer saline pH 7.4, 1 mM DTT and 1 mM EDTA. Fractions containing SAH hydrolase were pooled, the protein concentration determined and stored at xe2x88x9220xc2x0 C. until use.
The purity of the isolated enzyme was determined by SDS-polyacrylamidgradient gel electophoresis. From a 600 g weighty bovine kidney 30 mg pure SAH hydrolase could be prepared. The exchange of NAD+ by NADH results in a totally inactive enzyme.
Active SAH hydrolase can be inactivated by three different methods:
1. The tightly bound NAD+ of the active enzyme is removed by incubation with 150 mM NaCl, 8 mM ATP and 8 mM MgCl2 for 90 min at 37xc2x0 C. The enzyme solution was dialyzed and the resulted apo-enzyme is completely inactive and loses its binding affinity to adenosine. The reconstitution of the apo-enzyme with 1 mM NADH for 90 min at room temperature resulted in an enzymatically inactive enzyme with high adenosine binding capacity.
2. The enzymatically active enyzme is incubated with 2 volumes of saturated ammonium sulfate for 60 min at room temperature or at 0xc2x0 C. The solution is centrifuged at 12000xc3x97g for 30 min and the precipitate is dissolved in 20 mM TrisHCl pH 7.0 and dialyzed against the same buffer for 6 h. The apo-enzyme is reconstituted with 1 mM NADH for 90 min at room temperatur. The NADH-SAH hydrolase is enzymatically inactive, yet, its adenosine binding capacity is retained.
3. The enzymatically active enzyme is incubated with 100 xcexcM azido-adenosine at room temperature for 2 h. Thereafter the mixture is irradiated for 5 min using UV at 254 nm. The covalent photolabeling of azido-adenosine on SAH hydrolase results in a complete inactivation of the enzyme. The reduced SAH hydrolase retains its ability to bind adenosine with high capacity.
During the reduction procedure of SAH hydrolase, independent of the method used, about 25% of total protein (corresponding 7.5 mg) gets lost. The inactive ezyme (2 mg/ml) is stable at 4xc2x0 C. in 20 mM Tris, 40 mM Hepes pH 7.0 for at least 4 weeks and at xe2x88x9220xc2x0 C. for at least 2 month. Lyophylized enzyme (2 mg/ml) is stable at xe2x88x9220xc2x0 C. for 2 month. The production of inactive SAH hydrolase from one bovine kidney is sufficient to carry out 20000 measurements of adenosine.
Quantification of Adenosine by Displacement from its Binding Site
The adenosine concentration is determined by displacement experiments. The adenosine of the samples displaces the radioactive adenosine from the binding site of the reduced SAH hydrolase molecule.
The displacement of radioactive adenosine is performed in a final assay volume of 300 xcexcl containing the components as follows:
100 xcexcl reduced SAH hydrolase (1 xcexcg/300 xcexcl)
50 xcexcl 3H-adenosine (1 pmol/300 xcexcl) specific activity 54.5 Ci/mmol
100 xcexcl sample
50 xcexcl bufferxe2x80x9420 mM Tris, 40 mM Hepes pH 7.0
The equilibrium of adenosine binding to reduced enzyme is reached at room temperature after 10 h. Therefore the samples are incubated over night. The assay mixture is separated through nitrocellulose filters with a filtration equipment. The radioactive adenosine-enzyme-complex absorbed on the filters is determined by liquid scintillation counting.
To construct a standard curve, the log values of the adenosine concentrations are plotted against the 3H-adenosine bound (%) and the data resulted in a typical sigmoidal concentration-response curve. The adenosine values between 10xe2x88x929 and 10xe2x88x927 mol/l from the resulting curve is then used to calculate the adenosine values of samples on the basis of their counts per minutes observed.
Since adenosine has a high affinity to the inactive enzyme and radioactive 3H-adenosine is used as tracer according to the invention the detection limit for adenosine in this assay is 10xe2x88x929 mol/l or 3 pmol/sample.
This assay is an attractive alternative to HPLC in routine and research laboratories when only small sample volumes are available and a high sensitivity of the method is necessary. Further application of this analytical method are radio-contrast media examinations and renal allographs transplantation as a diagnostic tool to monitor renal function in transplanted patients.